1. Field of the Invention
The present invention relates to an isolated and cloned DNA sequence element that can be incorporated into expression vectors for the purpose of improving translation of a given mRNA and to enable the translation of the mRNA in a cap-independent manner.
2. Description of Related Art
There is an extensive and growing need to produce eukaryotic gene products in eukaryotic cells. Methods are needed to maximize expression of a desired gene and production of the gene product in eukaryotic cells. Initiation of eukaryotic protein synthesis involves about ten initiation factors, eIFs. The eIF4 group of initiation factors collectively catalyze the recognition of the mRNA cap, the unwinding of mRNA secondary structure, and the binding of mRNA to the 43S preinitiation complex [Pain, 1996]. The selection of a particular mRNA from the pool of translatable RNAs is determined by the relative efficiency of translation initiation by ribosome scanning and is largely governed by the composition and structure of the 5xe2x80x2-untranslated region (5xe2x80x2UTR) of the mRNA [Kozak, 1991; Sonenberg, 1996].
In certain instances translation of the transcribed, processed, and end-modified (i.e. capped and polyadenylated) mRNA is the limiting step in the production of the protein [Mathews et al., 1996; Meyuhas et al., 1996]. Translation is initiated by mRNA-protein interactions preceding the engagement of the small ribosomal subunit (40S) with the mRNA. Thus, limited availability of cap-binding proteins and competition with other cellular mRNAs for that proteins can be a rate-limiting factor in translation of the desired protein. This limitation can be even more pronounced under stress conditions (e.g. heat shock, hypoxia, nutrient deprivation) in which cap-dependent translation is markedly compromised. [Mathews et al., 1996; Meyuhas et al., 1996].
An alternative mode of translation is one in which ribosomes bind to the mRNA independent of the cap structure using an internal ribosome entry site (IRES), a specialized sequence within the 5xe2x80x2 untranslated regions that directly promote ribosome binding, independent of a cap structure. IRES elements were first discovered in picornaviral mRNAs which ate naturally uncapped but nonetheless efficiently translated [Jang et al., 1988; Pelletier and Sonenberg, 1988; Oh and Sarnow, 1995]. Subsequently, it was found that some cellular RNAs which are normally capped can be translated either by the 5xe2x80x2 end-dependent scanning mechanism or by an internal ribosome binding mechanism. Generally, IRES cannot be identified by sequence homology; known IRES have been identified and defined functionally [Mountford and Smith, 1995]. It appears that it is the conformation of the IRES sequence that enables the binding on the ribosome.
The list of cellular genes shown to contain sequences mediating internal initiation within their 5xe2x80x2UTR includes the immunoglubulin heavy chain binding protein (BiP) [Macjak and Sarnow, 1991], anntennapedia [OH et al., 1992], fibroblast growth factor (FGF) [Vagner et al., 1995], platelet-derived growth factor-B (PDGF-B) [Bernstein et al., 1997], insulin-like growth factor II (IGF-II) [Teerink et al., 1995], and the translation initiation factor eIF4G [Gan and Rhoads, 1996]. The potential utility of a cap-independent translation mode is best demonstrated in the case of viral RNAs in circumstances where cap-dependent translation is completely abrogated (through cleavage of an essential cap-binding protein by a virus-encoded protease) and the translation machinery is taken-over by IRES-containing viral RNA [Pelletier and Sonenberg, 1988]. The option of internal initiation is an advantage for competition with other mRNAs when certain components of the eIF4 complex become rate-limiting and this option provides a given mRNA the ability to be translated at times when cap-dependent translation is compromised. Such circumstances may develop under hypoxia where overall protein synthesis is significantly inhibited [Heacock and Sutherland, 1988; Kraggerud et al., 1995] or other stress conditions.
It would be useful to have a small sequence element, derived from a naturally-occurring cellular 5xe2x80x2UTR, that endows any desired gene with the ability to be more efficiently translated, in general, and to be translated in a cap-independent manner, in particular. That is, it would be useful to have additional IRES sequences with high translational efficiency, to use in expression vectors, to control mRNA translation and therefore protein synthesis as well as in gene therapy vectors.
According to the present invention, an isolated and cloned translation control element, and analogues thereof, having the nucleotide sequence as set forth in SEQ ID No:7 and designated SP163, is disclosed. The translation control element controls cap-independent mRNA translation via an internal ribosome entry site (IRES). The present invention provides expression vectors comprising the translation control element SP163 or its analogues operatively linked to a gene sequence to be expressed. In alternative embodiments, the expression vector comprises at least two nucleic acid sequences to be translated and SP163 is operatively linked to at least one of the sequences to be translated. The sequences to be translated may be linked to only one promoter in an embodiment.
The present invention provides a method for facilitating and enhancing cap-independent translation of mRNA by including in an expression cassette a translation control element having the nucleotide sequence as set forth in SEQ ID No:7 and designated SP163.
The novel sequence element (designated SP163) is composed of sequences derived from the 5xe2x80x2-UTR of VEGF (Vascular Endothelial Growth Factor gene), however, in a novel arrangement that was presumably generated through a previously unknown mode of alternative splicing. Functional analysis has shown that SP163 functions as a significant stimulator of translation and as a mediator of cap-independent translation. Imterestingly, the full-length 5xe2x80x2-UTR of VEGF has fair IRES activity indicating that these stronger activities were generated by a specific molecular event causing the juxtaposition of two specific 5xe2x80x2-UTR segments.
The advantages of SP163 is that it is a natural cellular IRES element with a superior performance as a translation stimulator and as a mediator of cap-independent translation relative to known cellular IRES elements. Another advantage of SP163 is that these functions are maintained under stress conditions.